Much interest has been focused on reactions for coupling nucleotides to form polynucleotide chains, and various chemical schemes have been described for the synthesis of polynucleotides. Typically these methods use a nucleoside reagent of the formula:
in which:
A represents H, OH, halogen, or an optionally protected hydroxyl group;
B is a purine or pyrimidine base whose exocyclic amine functional group is optionally protected;
one of M or Q is a conventional protective group for the 3′ or 5′—OH functional group while the other is:

where x may be 0 or 1, provided that:
a) when x=1:    R′ represents H and R″ represents a negatively charged oxygen atom; or    R′ is an oxygen atom and R″ represents either an oxygen atom or an oxygen atom carrying a protecting group; and
b) when x=0, R′ is an oxygen atom carrying a protecting group and R″ is either a hydrogen or a di-substituted amine group.
When x is equal to 1, R′ is an oxygen atom and R″ is an oxygen atom, the method is in this case the so-called phosphodiester method; when R″ is an oxygen atom carrying a protecting group, the method is in this case the so-called phosphotriester method.
When x is equal to 1, R′ is a hydrogen atom and R″ is a negatively charged oxygen atom, the method is known as the H-phosphonate method.
When x is equal to 0, R′ is an oxygen atom carrying a protecting group and R″ is a halogen, the method is known as the phosphite method, and when R″ is a leaving group of the disubstituted amine type, the method is known as the phosphoramidite method.
The conventional sequence used to prepare an oligonucleotide using reagents of the type of formula (I), basically follows four separate steps: (a) coupling a selected nucleoside which also has a protected hydroxy group, through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e., the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (“deprotection”) from the now substrate-bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate-bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, methyl amine, ethanolamine, or others, in a known manner.
The foregoing methods of preparing polynucleotides are well known and described in detail, for example, in Caruthers, Science 230: 281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310: 105-110, 1984; and in “Synthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivatives”, CRC Press, Boca Raton, Fla. pages 100 et seq., U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 5,153,319, 5,869,643, EP 0294196, and elsewhere. The phosphoramidite and phosphite triester approaches are most broadly used, but other approaches include the phosphodiester approach, the phosphotriester approach and the H-phosphonate approach. Such approaches are described in Beaucage et al., Tetrahedron (1992) 12:2223-2311. A more recent approach for synthesis of polynucleotides is described in U.S. Pat. No. 6,222,030 B1 to Dellinger et al, Issued Apr. 24, 2001.
In the typical phosphoramidite method of solid phase oligonucleotide synthesis, the synthesis typically proceeds in the 3′ to 5′ direction (referring to the sugar component of the added nucleoside), although the synthesis may easily be conducted in the reverse direction. The added nucleoside generally has a dimethoxytrityl protecting group on its 5′ hydroxyl and a phosphoramidite functionality on its 3′ hydroxyl position. Beaucage et al. (1981) Tetrahedron Lett. 22:1859. See FIG. 1 for a schematic representation of this technology. In FIG. 1 “B” represents a purine or pyrimidine base, “DMT” represents dimethoxytrityl protecting group and “iPr” represents isopropyl. In the first step of the synthesis cycle, the “coupling” step, the 5′ end of the growing chain is coupled with the 3′ phosphoramidite of the incoming monomer to form a phosphite triester intermediate (the 5′ hydroxyl protecting group prevents more than one monomer per synthesis cycle from attaching to the growing chain). Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185. Next, the optional “capping reaction” is used to stop the synthesis on any chains having an unreacted 5′ hydroxyl, which would be one nucleotide short at the end of synthesis. The “capping reaction” has the added benefit of cleaving coupling products from the heterobases, such as O-6 adducts on guanosine, prior to the oxidation step. The phosphite triester intermediate is subjected to oxidation (the “oxidation” step) after each coupling reaction to yield a more stable phosphotriester intermediate. Without oxidation, the unstable phosphite triester linkage would cleave under the acidic conditions of subsequent synthesis steps. Letsinger et al. (1976) J. Am. Chem. Soc. 98:3655. Removal of the 5′ protecting group of the newly added monomer (the “deprotection” step) is typically accomplished by reaction with acidic solution to yield a free 5′ hydroxyl group, which can be coupled to the next protected nucleoside phosphoramidite. This process is repeated for each monomer added until the desired sequence is synthesized.
According to some protocols, the synthesis cycle of couple, cap, oxidize, and deprotect is shortened by omitting the capping step or by taking the oxidation step ‘outside’ of the cycle and performing a single oxidation reaction on the completed chain. For example, oligonucleotide synthesis according to H-phosphonate protocols will permit a single oxidation step at the conclusion of the synthesis cycles. However, coupling yields are less efficient than those for phosphoramidite chemistry and oxidation requires longer times and harsher reagents than amidite chemistry.
Conventional synthesis protocols of oligonucleotides are not without disadvantages. For example, cleavage of the DMT protecting group under acidic conditions gives rise to the resonance-stabilized and long-lived bis(p-anisyl)phenylmethyl carbocation. Gilham et al. (1959) J. Am. Chem. Soc. 81:4647. Protection and deprotection of hydroxyl groups with DMT are thus readily reversible reactions, resulting in incomplete reactions during oligonucleotide synthesis giving rise to sequence deletions and a lower yield than might otherwise be obtained. To circumvent such problems, large excesses of acid are used with DMT to achieve quantitative deprotection. However, the repeated exposure of DNA sequences to acids gives rise to acid catalyzed removal of the heterobases from the sugar ring. Heterobase removal is most facile on the purine bases, adenosine, and guanosine; resulting in depurination, however all heterobases and modified heterobases can be susceptible to acid catalyzed removal. As bed volume of the polymer is increased in larger scale synthesis, increasingly greater quantities of acid are required. The acid-catalyzed depurination that occurs during the synthesis of oligodeoxyribonucleotides is thus increased by the scale of synthesis. Caruthers et al., in Genetic Engineering: Principles and Methods, J. K. Setlow et al., Eds. (New York: Plenum Press, 1982). Solvent use in larger scale synthesis becomes increasingly prohibitive as well, as more washing is required. In particular, the reagents used in the coupling step typically are highly susceptible to hydrolysis, which requires dry solvents, further increasing the cost of solvents.